Self assembly of sensor membranes

ABSTRACT

Electrode membrane combinations for use in biosensors to detect analytes in a sample and methods for making and storing same are disclosed. In one aspect, a method is provided for producing a first layer electrode membrane comprising: 
     (1) Forming a solution containing Linker Lipid  A , the disulfide of mercaptoacetic acid (MAAD) or similar molecule, linker Gramicidin B, membrane spanning lipid C (MSL-C) and membrane spanning lipid D (MSL-D) or other suitable linker molecules and other ion channel combinations; 
     (2) Contacting an electrode containing a clean gold surface with the solution, the disulfide containing components in the solution thus adsorbing onto the gold surface of the electrode; 
     (3) Rinsing the electrode with a suitable organic solvent; and 
     (4) Removing the excess organic solvent used for rinsing.

This is a divisional of Ser. No. 08/685,329 filed on Jul. 23, 1986 nowU.S. Pat. No. 5,879,878 which is 371 PCT/AU96/00639 filed on Jun. 20,1996.

The present invention relates to electrode membrane combinations for usein biosensors to detect analytes in a sample and to methods for theproduction of such electrode membrane combinations. The presentinvention also relates to methods for storing such electrode membranecombinations.

Biosensors based on ion channels or ionophores contained within lipidmembranes that are deposited onto metal electrodes and where the ionchannels are switched in the presence of analyte molecules have beendescribed in International patent specification Nos WO 92/17788, WO93/21528, WO 94/07593 and U.S. Pat. No. 5,204,239 (the disclosures ofwhich are incorporated herein by reference). As is disclosed in theseapplications, ionophores such as gramicidin ion channels may beco-dispersed with amphiphilic molecules, thereby forming lipid membraneswith altered properties in relation to the permeability of ions. Thereis also disclosure of various methods of gating these ion channels (forexample, the lateral segregation mechanism disclosed in InternationalPatent Application WO90/08783) such that in response to the binding ofan analyte to a binding partner attached to the membrane, theconductivity of the membrane is altered. The applications also disclosemethods of producing membranes with improved sensitivity using a surfaceamplifier effect, and improved stability and ion flux using chemisorbedarrays of amphiphilic molecules attached to an electrode surface. Theapplications further disclose means of producing lipid membranesincorporating ionophores on said chemisorbed amphiphilic molecules.

The present inventors have now determined improved means of producingelectrode membrane combinations that result in sensor membranes withimproved properties in terms of reproducibility, gating response towardsan analyte, lateral segregation response, surface amplifier effect,stability in serum, plasma and blood, simplified production and theability to store the membranes in a dry format (i.e. in the absence ofany aqueous bath solution).

In the first aspect, the present invention consists in a method ofproducing a first layer electrode membrane comprising:

(1) Forming a solution containing Linker Lipid A (FIG. 1), the disulfideof mercaptoacetic acid (MAAD) or similar molecule, such as EDS linkerGramicidin B (FIG. 2), membrane spanning lipid C (MSL-C) (FIG. 3) andmembrane spanning lipid D (MSL-D) (FIG. 3) or other linker molecules andion channel or ionophore combinations as previously described;

(2) Contacting an electrode containing a clean gold surface with thesolution, the disulfide containing components in the solution thusadsorbing onto the gold surface of the electrode;

(3) Rinsing the electrode with a suitable organic solvent; and

(4) Removing the excess organic solvent used for rinsing.

The nature of the membrane components are as follows:

Linker Lipid A comprising a benzyl disulfide attachment region, ahydrophilic region composed, in sequence, of tetraethylene glycol,succinic acid, tetraethylene glycol and succinic acid subgroups and analiphatic chain;

The disulfide of mercaptoacetic acid (MAAD) or similar molecule, such asthe disulfide of 2-mercaptoethanol (EDS).

Linker Gramicidin B is a linker molecule which comprises a benzyldisulfide attachment region, a hydrophilic region composed, in sequence,of tetraethylene glycol, succinic acid, tetraethylene glycol, succinicacid, and a hydrophobic region of gramicidin;

Membrane spanning lipid (MSL) D which comprises a benzyl disulfideattachment region, a hydrophilic region composed, in sequence, oftetraethylene glycol, succinic acid, tetraethylene glycol, succinic acidand a hydrophobic region of 1,1′dotriacontamethylenebis (2-3 RS,7R,11-phytanyl) with an intermediate biphenyl region and a head group ofphosphatidylcholine, hydroxyl, succinic acid, or PEG-400 COOH; and

Membrane spanning lipid C which comprises the same attachment andhydrophilic region as membrane spanning lipid D but differs in the headgroup which is a group consisting of (one to eight) 1,6-amino caproicacid and biotin.

In a preferred embodiment of the present invention the ratio of LinkerLipid A to the disulfide of mercaptoacetic acid (MAAD) or2-mercaptoethoethanol (EDS) is 5:1 to 1:2, more preferably is 2:1.

It is further preferred that in order to improve the stability of themembrane, the amount of MSL-D in the first layer is as high as can beallowed and still maintain reasonable gramicidin conduction. The ratioof (Linker Lipid A+MAAD or EDS) to MSL-D is therefore preferably between10:1 to 100:1.

In a further preferred embodiment, the amount of MSL-C is such that inthe final sensor membrane an effective surface amplification on additionof analyte occurs, while still making it possible to suppress thelateral segregation induced gating on addition of the streptavidin,avidin or other similar biotin-binding protein. It should be noted thatif the amount of MSL-C in the final sensor membrane is too large, thenthe excess protein that is bound to the MSL-C on addition of thestreptavidin, avidin or similar biotin-binding protein will restrict themobility of the gramicidin/receptor couple thereby reducing the gatingresponse. In cases where the analyte molecule has multiple identicalepitopes, MSL-C may capture the analyte molecules in preference togramicidin/receptor couple, reducing the biosensor response.

It is therefore preferred that the ratio of (Linker Lipid A+MAAD or EDS)to membrane spanning lipid C is between 20,000:1 and 100:1.

It is further preferred that the ratio of (Linker Lipid A+MAAD or EDS)to MSL-C is 20,000:1.

As is known in the art, gramicidin exists in a monomer/dimer equilibriumin a bilayer membrane. In order for the gramicidin lateral segregationswitch to function effectively, the ratio of monomer to dimer must becontrolled. It is preferred that a proportion of the gramicidin ionchannels exist as freely diffusing monomers in the outer membrane layer.The ratio of monomers to dimers can be controlled, amongst othermethods, by changing the concentration of gramicidin in the first andsecond half of the membrane.

It is therefore preferred that the ratio of (Linker Lipid A+MAAD or EDS)to linker Gramicidin B is 10,000:1.

It is further preferred that the ratio of (Linker Lipid A+MAAD or EDS)to linker Gramicidin B is between 20,000:1 and 100,000:1 in those caseswhere it is necessary to minimise the amount of background leakage dueto the adsorbed linker Gramicidin B.

It is preferred that the gold electrode consists of a freshly evaporatedor sputtered gold electrode. It is further preferred that the goldelectrode surface be freshly cleaned using a plasma etching process oran ion beam milling process.

It is preferred that the solvent for the adsorbing solution (step (1)and for the rinsing step (4) is ethanol.

In a second aspect, the present invention consists in a method ofproducing a monolayer electrode membrane comprising:

(1) Forming a solution containing the disulfide of mercaptoacetic acid(MAAD) or similar molecule (e.g. 2-mercaptoethanol (EDS)), membranespanning lipid C(MSL-C) and/or membrane spanning lipid D (MSL-D) and,optionally, Linker Lipid A, linker Gramicidin B or other linkermolecules or ion channel or ionophore combinations;

(2) Contacting an electrode containing a clean gold surface with thesolution, the disulfide containing components in a solution thusadsorbing onto the gold surface of the electrode;

(3) Rinsing the electrode with a suitable organic solvent; and

(4) Removing the excess organic solvent used for rinsing,

wherein the solution in step (1) contains more than a molar % of 50% ofa membrane spanning lipid.

More preferably, the solution in step (1) contains more than a molar %or 70% of a membrane spanning lipid, 29% MAAD or EDS and 1% othermembrane spanning lipids.

The preferred features and embodiments discussed above in regard to themethod of the first aspect of the invention, may be equally applicableto the method of the second aspect of the invention.

The membranes produced by the method of the second aspect of theinvention, do not form bilayers and have been found to be particularlyresistant towards non-specific effects on addition of serum, plasma orwhole blood to the sensor. Further advantages have been noted in thatthese membranes may be reused over a period of months in serum, plasmaor whole blood without showing signs of degradation of performance.Monolayer lipid membranes are more practical for manufacturing purposes,have fewer manufacturing steps and greater stability, leading to a laterexpiry on the manufactured sensor containing such membranes. In this itis also preferable for the spacer molecule, MAAD or EDS, to becovalently linked to the membrane spanning lipids C or D, and covalentlylinked to PEPC, GDPE or triphytanyl PC, which increases stability of thefinal membrane.

A further preferred embodiment of the method according to the secondaspect of the invention, consists in the use of valinomycin, covalentlylinked to the membrane spanning lipids C or D, via a linker ofappropriate length such that the valinomycin is able to diffuse from oneside of the membrane to another. This then results in a reusablebiosensor, which does not need replenishment of the ionophore and couldbe used for an implantable device.

The present inventors have determined that the production of thebiosensor is simplified and improved through the use of streptavidin,avidin or one of the related biotin binding—proteins as a means ofcoupling a biotinylated receptor onto a biotinylated gramicidin ionchannel or MSL.

In a third aspect, the present invention consists in a method ofproducing a second layer electrode membrane combination utilisingbiotinylated gramicidin E, in which the biotin is attached to thegramicidin via an amide to a lysine residue (preferred for chemicalstability) or via an ester link to ethanolamine using a linker arm thatis made up of between 1 to 8 aminocaproyl groups. The linker length,type, valency and number of linkers can affect the stability of thecompleted sensor and the optimum linker varies depending on the analytebeing measured. The method comprises:

(1) Adding a solution of lipid and biotinylated gramicidin E (FIG. 4),dispersed in a suitable solvent onto the electrode surface containing afirst layer produced as described in the first aspect of the presentinvention;

(2) Rinsing the electrode surface with an aqueous solution:

(3) Adding a solution of streptavidin, avidin, neutravidin, avidin orstreptavidin derivative;

(4) Rinsing the electrode with an aqueous solution in order to removeexcess streptavidin, avidin, neutravidin or other avidin or streptavidinderivative;

(5) Adding a solution of a biotinylated binding partner molecule; and

(6) Rinsing the coated electrode with an aqueous solution.

In a preferred embodiment of the present invention the lipid used instep (1) of the method of the third aspect is a mixture of diphytanylphosphatidyl choline and glyceryl diphytanyl ether. The inventors havefound that the combination of these lipids improves the stability of thebilayer membrane towards serum, plasma and whole blood, while stillmaintaining a good ionic seal, fluidity, reducing temperature effects onconduction and maintaining a true bilayer membrane structure.

It is further preferred that the diphytanyl phosphatidyl choline (DPEPC)and glyceryl diphytanyl ether (GDPE) is in a 7:3 ratio.

It is further preferred that the lipid is a triphytanyl phosphorylcholine as shown in FIG. (6).

It is also preferred that membranes contain 0 to 50%, more preferably 0to 20% cholesterol in the second layer to enhance stability and analyteresponse in a serum, plasma or whole blood sample.

It is preferred that the ratio of lipid to biotinylated gramicidin E isbetween 10,000:1 and 1,000,000:1.

It is further preferred that the ratio of lipid to biotinylatedgramicidin E is 100,000:1.

It is preferred that the biotin is attached to the gramicidin via theethanolamine end using a linker arm that is between 10-80 angstromslong. It is preferred that the linker arm is hydrophilic.

It is preferred that the biotin is attached to the gramicidin via theethanolamine end using a linker arm that is made up of between 1 to 8aminocaproyl groups.

It is further preferred that two biotins are attached to the gramicidinvia the ethanolamine end such that the biotins are able to bindsimultaneously into the adjacent binding sites of one streptavidin,avidin or similar biotin-binding protein molecule, or into two separatestreptavidin avidin or similar biotin-binding protein molecules.Alternatively, more than two biotin molecules can be attached to thegramicidin to produce multiple attachment sites for the binding partnermolecules.

It is preferred that the two biotins are attached to the gramicidin viathe ethanolamine end such that each biotin is attached to two to fourlinearly joined aminocaproyl groups that are attached to a lysine groupas shown in FIG. (5). When more than two biotin molecules are attachedto the gramicidin, a longer linker up to twenty aminocaproyl groups maybe necessary these may be organised linearly or as a branched structure.

It is further preferred that in order to optimise the analyte response,it is necessary to minimise the signal caused by the presence of thelinker. Thus, the amount of streptavidin, avidin or other similarbiotin-binding protein that is added in step (3) is sufficient to causea prozone effect, allowing most of the available biotinylated species inthe membrane to have one streptavidin or related molecule bound toprevent crosslinking between gramicidin channels and MSL until a samplecontaining analyte is added to the sensor.

It is further preferred that prior to the addition of the streptavidin,avidin, or similar biotin-binding protein the lipid membrane electrodeassembly is cooled. This reduces the fluidity of the membrane,decreasing the mobility of membrane components thus allowing thestreptavidin, avidin or other similar biotin-binding protein to morereadily bind to the biotinylated Gramicidin E and the membrane spanninglipid C without crosslinking between gramicidin channels and MSL until asample containing analyte is added to the sensor.

It is preferred that the lipid membrane electrode is cooled to between0° and 50° C., more preferably 0° and 5° C. It is further preferred thatthe subsequent rinsing and addition of the biotinylated binding partnermolecule are also carried out at 0° to 50° C., more preferably 0° to 5°C.

It is preferred that the binding partner molecule is a biotinylatedantibody or biotinylated antibody fragment.

It is further preferred that the binding partner molecule is a Fab′fragment that is biotinylated via the free Fab′ thiol group.

Is further preferred that the linker between the Fab′ and biotins isbetween 10-80 angstroms in length. Is further preferred that the linkerbetween the Fab′ and biotins consists of one to eight aminocaproylgroups.

It is further preferred that the group containing two biotins isattached to the antibody or antibody fragment such that the two biotinsare able to complex simultaneously one streptavidin, avidin or othersimilar biotin-binding protein or two adjacent streptavidin, avidin orother similar biotin-binding protein molecules.

Alternatively, more than two biotins may be attached to the antibody orantibody fragment.

Furthermore, the present inventors have determined that by producing acovalently or passively coupled conjugate between the binding partnermolecule and the streptavidin, avidin or other similar biotin bindingprotein the production of the biosensor membrane is further simplified.

Accordingly, steps 3 to 5 of the method of the third aspect can besubstituted with:

(3) Adding a solution containing a conjugate between streptavidin,avidin, neutravidin or other avidin or streptavidin derivative and amolecule which is a member of a binding pair.

It is preferred that the binding partner molecule is an antibody or anantibody fragment such as an Fab or Fab′ or Fv fragment. Other bindingpairs, which could be used in this invention would include: naturallyoccurring binding proteins and cellular receptors/analytes, enzymes orenzyme analogues/substrates, lectins/carbohydrates, complementarynucleic acid sequences and Anti-FC, Protein A or Protein G/antibody.

In order to manufacture sensor membranes efficiently and reproducibly,it is advantageous to incorporate the ionophore separate to theassembled membrane. It is also advantageous to bind one binding partnerto the ionophore, before incorporation into the membrane. This bothcontrols and enhances the reproducibility of membrane conduction andallows the reproducible attachment of the second binding partner neededin a two site immuno- or similar assay system, ensuring that only thefirst binding partner is attached to ionophore and only the secondbinding partner is attached to a second ionophore or MSL.

The present inventors have found that it is possible to co-disperse thehydrophobic ionophore in aqueous solution by several means, including:

1. The presence of a detergent, preferably at levels below the criticalmicelle concentration of the detergent, such that ionophore and thedetergent form aggregates which allow the ionophore to remain insolution;

2. Conjugation of the gramicidin or other ionophore to a large molecularweight water soluble species; and

3. Attachment of the ionophore to a bead.

Furthermore it was found that it was possible to incorporate thefunctional ionophore into the biosensor lipid membranes by adding anaqueous solution of the ionophore/detergent aggregate to the solutionbathing the preformed lipid biosensor membrane. This method of additionof the ionophore allows for a more controlled and reproducible method ofincorporation of the ionophore into the lipid membrane.

Accordingly, in a fourth aspect, the present invention consists in amethod of producing a second layer electrode membrane combinationcomprising:

(1) Adding a solution of lipid dispersed in a suitable solvent onto theelectrode surface containing a first layer produced as described in themethod of the first aspect of the present invention;

(2) Rinsing the electrode surface with an aqueous solution;

(3) Adding an aqueous solution containing ionophore co-dispersed withdetergent or solubilised by coupling to a high molecular weight solublespecies;

(4) Rinsing the electrode with an aqueous solution; and

(5) Adding the receptor using either streptavidin, avidin, or othersimilar biotin-binding protein followed by addition of a biotinylatedantibody or antibody fragment or adding a streptavidin, avidin orsimilar biotin-binding protein conjugated to an antibody or antibodyfragment as detailed in the third aspect of the present invention.

In a preferred embodiment of the present invention the lipid used instep (1) is a mixture of diphytanyl phosphatidyl choline and glyceryldiphytanyl ether. It has been found that the combination of these lipidsimproves the stability of the bilayer membrane towards serum, plasma andwhole blood, while still maintaining a good ionic seal, fluidity,reducing temperature effects on conduction and maintaining a truebilayer membrane structure.

It is further preferred that the diphytanyl phosphatidyl choline andglyceryl diphytanyl ether is in a 7:3 ratio.

It is further preferred that the lipid is a triphytanyl phosphorylcholine as shown in FIG. (6).

It is also preferred that membranes contain 0 to 50%, more preferably upto 20% cholesterol in the second layer to enhance stability and analyteresponse in a serum, plasma or whole blood sample.

It is preferred that the aqueous solution used in step (3) of the fourthaspect of the present invention, contains gramicidin or a gramicidinderivative that is added to an aqueous solution of a detergent such thatthe detergent is present in excess relative to the gramicidin but thatthe total concentration of detergent is below the critical micelleconcentration (CMC). The total detergent concentration is preferablykept below the CMC in order to minimise or negate any possibledisruption of the membrane by the detergent.

The gramicidin/detergent solution is then preferably sonicated using anultrasonic bath or horn for 5 to 20 minutes.

Preferred detergents are sodium dodecylsulfate, octylglucoside, tween,or other ionic or non-ionic detergents.

It is further preferred that the alkyl chain contains at least 7 or moremethylene groups.

It is preferred that the detergent is sodium dodecylsulfate.

It is further preferred that the concentration of the sodiumdodecylsulfate is less than 0.00001M and that the concentration ofgramicidin is ten times less than the sodium dodecylsulfateconcentration.

It has been found that, if it is necessary to store the electrodes whichalready have the first layer of the membrane adsorbed onto the electrodesurface, then it is advantageous to store said electrodes covered in asolvent. This method of storing the electrode with the first layermembrane in a solution has been found to produce subsequent sensormembranes with improved homogeneity and ionophore gating ability,compared with storing the electrode in air.

Accordingly, in a fifth aspect the present invention consists in a firstlayer membrane electrode combination comprising an electrode and a firstlayer membrane comprising a closely packed array of amphiphilicmolecules and a plurality of ionophores, the first layer membrane beingconnected to the electrode by means of a linker group as describedpreviously, said first layer membrane being stored in the presence of asolvent.

Electrodes may be stored in a gaseous or liquid environment and, in apreferred embodiment, the solvent in which the electrodes are stored isan organic solvent or an aqueous solvent.

If the solvent is an organic solvent, it is further preferred that thesolvent is an alcohol such as ethanol, glycerol, ethylene glycol, analcohol or diol containing between 3 to 12 carbon atoms.

It is further preferred that the solvent is a hydrocarbon with between 8to 20 carbon atoms. It is further preferred that the solvent is anaqueous solution containing a detergent.

It is further preferred that the solvent is a compound that is able tocoat the electrodes such that oxidation of the electrode surface isminimised. It is preferred that such a solvent can be applied as a thinfilm.

Additionally it has been found that it is possible to store the completesensor membrane electrode combination in a non-aqueous format. This ishighly advantageous in terms of ease of manufacturing, shipping andstoring of the biosensor product.

Accordingly, in a sixth aspect, the present invention consists in alipid membrane based biosensor comprising a lipid membrane incorporatingionophores, the conductivity of the membrane being dependent on thepresence or absence of an analyte, wherein the aqueous bathing solutionin which the biosensor normally resides, is removed in a manner suchthat, on drying of said lipid membrane biosensor, the lipid membrane andthe receptor molecules retain their function, structure and activity,when rehydrated.

It is preferred that in the drying process that the biosensor membranedoes not have contact with the air-water interface, hence methods ofdrying such as lyophilisation, evaporation, or evaporation overcontrolled humidity, are recommended. It is also preferred that theconcentration of the water-replacing agent is sufficient to protect allcomponents within the membrane, i.e. lipid, ionophore and protein,during the drying process, during the storage time, and yet is easilyremoved upon the first addition of analyte or sample in the appropriatematrix, such that full activity of the biosensor membrane is restoredimmediately.

The water replacing substance may be either a protein, a low molecularweight diol or triol, a polyethylene glycol, a low molecular weightsugar, a polymeric peptide, polyelectrolyte or combinations of thesesubstances, all of which are well known in the art. The main attributesof the water substitute are that it is highly polar, has a low vapourpressure, allows the membrane to retain its structure, is proteincompatible and does not impede biosensor function when rehydrated. Thesesubstances may also be covalently bound to a specific membranecomponent, preferably a membrane spanning lipid.

It is preferred that the water replacing substance is bovine serumalbumin, serum, fish gelatin, non-fat dry milk powder, casein, glycerol,ethylene glycol, diethylene glycol, polyethylene glycol, trehalose,xylose, glucose, sucrose, dextrose, ficoll and it is further preferredthat the water replacing molecule is glycerol, sucrose, dextran ortrehalose.

Such classes of molecules may also have the additional advantage in thebiosensor to act as a spreading layer for serum/blood/analyte fluidaddition; as a filter against specific cells, bacteria, virus particles,or classes of molecules; or as a reservoir containing specificdisplacement reagents required to compete off small analytes fromproteins to which they are bound in serum or blood.

A further advantage of the water substituting agent is that it allowsfor the controlled rehydration of the lipid membrane without the lipidbilayer being in contact with the air/water interface as the analytesolution or sample is added.

An example of the latter is given in FIG. 13, where water-replacingmolecules are either added or covalently bound to regions of themembrane and contain, for example, ANS (8-anilino-1-naphthalene-sulfonicacid) which competes with thyroxine for binding sites in albumin andthyroxine-binding globulin (TBG), releasing thyroxine for subsequentdetection by the biosensor membrane.”

The invention is hereinafter further described with reference to thefollowing non-limiting examples and accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows Linker Lipid A which comprises a disulfide attachmentregion, a hydrophilic region composed (in sequence) of tetraethyleneglycol, succinic acid, tetraethylene glycol, succinic acid subgroups andan aliphatic chain.

FIG. 2 shows a linker molecule which comprises a benzyl disulfideattachment region, a hydrophilic region composed (in sequence) oftetraethylene glycol, succinic acid, tetraethylene glycol, succinicacid, and a hydrophobic region of gramicidin.

FIG. 3 shows membrane spanning lipids C and D. Membrane spanning lipid Cterminates in a group consisting of between one to eight 1,6-aminocaproic groups and biotin. Membrane spanning lipid D comprises the headgroup which is a group consisting of phosphatidylcholine (PC), OH,succinic acid or polyethylene glycol (PEG) 400.

FIG. 4 shows biotinylated gramicidin E in which the biotin is attachedto the gramicidin via the ethanolamine end using a linker arm that ismade up of between 1 to 8 aminocaproyl groups.

FIG. 5 shows two biotins attached to gramicidin via the ethanolamine endwith each biotin attached to two to four linearly joined aminocaproylgroups that are attached to a lysine group.

FIG. 6 shows a lipid consisting of a triphytanyl phosphoryl choline.

FIGS. 7a-7 d show the impedance change of the biosensor owing to K+ inhuman blood.

FIG. 8 shows repeated whole blood K+ detection in monolayer biosensormembrane.

FIG. 9 shows that the biosensor response in whole blood was a functionof the K+ concentration.

FIGS. 10a-10 f show the detection of ferritin in whole blood and serum.

FIG. 11 shows the response of the biosensor to serum concentrations offerritin.

FIG. 12 shows the dependence of the streptavidin response on linker typean length.

FIG. 13 shows a network of water-replacing agent (i.e. protectantsubstance) linked to the biosensor membrane, containing thyroxinedisplacement reagent such as ANS. As the ANS molecules compete forprotein binding sites, thyroxine is released and diffuses to themembrane where it is detected by competitive assay (thyroxine-gramicidinbound to anti-thyroxine Ab/Fab which is bound to membrane spanninglipids, is competed off with the released thyroxine, leading to therelease of gramicidin channels and return of ionic conduction in thebiosensor).

FIG. 14 shows the impedance spectra of a membrane prior to addition of“linker gramicidin E”/SDS (1); after the addition of “linker gramicidinE”/SDS and after addition of streptavidin and biotinylated anti-ferritinFab′ (2); and the spectrum after the addition of ferritin (3).

FIG. 15 shows the impedance spectra of a membrane prior to addition of“linker gramicidin F”/streptavidin (1); after the addition of “linkergramicidin F”/streptavidin and after the addition of biotinylatedanti-TSH Fab's (2); and the spectrum after the addition of TSH (3).

EXAMPLE 1

Whole Blood K+ Detection in Monolayer Biosensor Membranes

Ion carriers other than gramicidin may be used in the membranesdescribed in this invention. Simplified membranes formed mainly frommembrane spanning lipid components can form extremely robust andbiocompatible biosensors. Fewer membrane components also provides easiermanufacturability.

Monolayer membrane: 100 uM MSLPC (MSL-D) 20 uM MAAD

Electrodes with freshly evaporated gold (1000A) on chrome adhesion layer(200A on glass microscope slides) were dipped into an ethanol solutionof the above components, rinsed with ethanol, then stored at 4° C. underethanol until used for impedance measurements (days-weeks). The slidewas clamped into a blocked containing teflon coated wells which definedthe area of the working electrode as approximately 16 mm². PBS bufferwas added followed by 5 ul of 1 mM valinomycin in ethanol, an ionophorespecific for K+ detection. Impedance measurements were carried out.

FIG. 7 shows that the impedance change showing the biosensor response toK+ in human whole blood (obtained from a healthy volunteer) was specificto the presence of valinomycin incorporated into the biosensor membrane.The volume of PBS buffer in the electrode well was completely exchangedfor 100 ul whole blood which resulted in the decrease in impedance as K+ions in blood were transported across the biosensor membrane byvalinomycin. The addition of whole blood showed no detrimental effectsto the lipid membrane.

Even more convenient assembly of the membrane would be with membranecomponents which are covalently linker together and synthesised as asingle molecule. Thus a membrane spanning lipid can be linked to spacermolecules such as MAAD or its analogues (to provide correct packing ofthe first layer which enables essential ion channel or ionophorediffusion), and to extra hydrocarbon chains such as phytanyls to provideincreased fluidizing properties to the membrane spanning lipid.

EXAMPLE 2

Repeated Whole Blood K+ Detection in Same Monolayer Biosensor Membrane

The robust and biocompatible characteristics of the monolayer biosensorcan be illustrated by its ability to sustain repeated exposure to wholeblood and still function as a biosensor.

Monolayer membrane: 100 uM MSL-succinic acid (MSL-D) 20 uM MAAD

Electrodes were prepared and measured as described in Example 1. Afteraddition of valinomycin, 80 ul PBS buffer was removed from the electrodewell (160 ul initial volume) and replaced by 80 ul whole blood. Afterthe change in impedance due to the K+ response was recorded, the wellwas washed 4 times with PBS buffer and valinomycin was again added tothe well before the next exposure of whole blood. The cycle was repeated4 times as shown in FIG. 8, showing the biosensor remaining intact andfunctioning even after repeated exposure to whole blood.

EXAMPLE 3

Whole Blood K+ Titration in Biosensor Membranes

This example shows that the detection ability of the biosensor is withinthe clinically-relevant range of 3-6 mM, and above, if required. Thebiosensor is also stable to whole blood addition even in the bilayer,rather than monolayer, configuration.

1st layer: 100 uM MSL-PEG 400-COOH (MSL-D) 0.8 mM MAAD 1 mM DLP

2nd layer: 14 mM (C18DPEPC:C₁₈GMPE=7:3): vahinomycin=100:1

(Note: C₁₈DPEPC=DPEPC with 2 additional CH₂ groups; C₁₈GMPE=GDPE with amonophytanyl chain instead of diphytanyl chains and 2 additional CH₂groups).

Electrodes were prepared as described in Example 1. The 2nd layer wasadded from an ethanolic solution then PBS buffer was added, and theelectrode well was washed 4 times. Whole blood was added to differentelectrodes and the change of impedance with K+ concentration recorded.FIG. 9 shows that the biosensor response to K+ in human whole blood wasa function of the concentration of K+ in the blood. The addition ofwhole blood showed no detrimental effects to the lipid membrane.

EXAMPLE 4

Detection of Endogenous Ferritin in Whole Blood and Serum

The bilayer biosensor is stable to whole blood addition and is fullyfunctional for a single-step, endogenous ferritin detection inunprocessed blood. (Blood was obtained from a volunteer using CP2D asanticoagulant, which contains citric acid, sodium citrate, sodium acidphosphate, and dextrose).

1st layer: 9.3 nM Gayy (Linker B) 5.5 nM NMSLXXB (MSL-C) 1.1 uM MSLOH(MSL-D) 37 uM MAAD 75 uM DLP (Linker A)

2nd layer: 14 mM (DPEPC:GDPE=7:3): Ga5XB (Linker E)=100,000:1

Electrodes were prepared and 2nd layer added as described in Example 3.Ferritin in either PBS, serum or whole blood was added to differentelectrodes [FIGS. 10a), b), and c)], and the change of impedance withferritin concentration was recorded. FIG. 4 also shows the stepspreceding the addition of analyte, i.e. when SA is added (5 ul 1 mg/mlin PBS), washing out excess SA, then in FIGS. 10a), b), and c) only, theaddition of anti-ferritin Fab′ biotinylated at the thiol group (5 ul0.05 mg/ml in PBS) and the subsequent rinsing step, is also shown.

FIGS. 10d), e), and f) shows that in the absence of specific receptorsfor ferritin, there is no response, regardless of whether PBS, serum orwhole blood was added. Note that no other reagents or washing steps arerequired after the sample containing the analyte has been added. Thebiosensor is manufactured up to the stage where specific receptors areadded, then it is ready for the one-step addition of the sample.

EXAMPLE 5

Titration Curve from Endogenous Ferritin in Human Serum

After clotting, blood obtained from 4 different volunteers wascentrifuged down and serum was separated and used for the presentexample.

1st layer: 9.3 nM Gayy (Linker B) 5.5 nM MSLXXXXB (MSL-C) 1.1 uM MSLOH(MSL-D) 37 uM MAAD 75 uM DLP (Linker A)

2nd layer: 14 mM (DPEPC:GDPE=7:3): Ga5XB (Linker E)=66,667:1

Electrodes were prepared and 2nd layer added as described in Example 4.From the initial well volume of 150 ul, 100 ul buffer was removed andreplaced with 150 ul serum. Different sera were added to differentelectrodes and the tau (s) of the ferritin response was calculated fromthe admittance at minimum phase. FIG. 11 shows the response of thebiosensor to sera containing one of the following concentrations offerritin (measured using the Immulite autoanalyser); 18.8, 38, 280, or476 pM, in duplicate.

FIGS. 10d), e), and f) shows that in the absence of specific receptorsfor ferritin, there is no response, regardless of whether PBS, serum orwhole blood was added. Note that no other reagents or washing steps arerequired after the sample containing the analyte has been added. Thebiosensor is manufactured up to the stage where specific receptors areadded, then it is ready for the one-step addition of the sample.

EXAMPLE 6

Variation of Analyte Response with Gramicidin—Biotin Linker Length andType

The linker length between gramicidin and biotin can be varied by addingvarying numbers of caproyl groups, or by adding “multi-armed” linkers togramicidin, each linker terminating with a biotin molecule, thusenabling a single gramicidin to capture either two biotin sites withinthe same SA molecule, or two or more SA molecules, depending on thelength of the linker and number of “arms” present.

1st layer: 0.1 uM Gayy (Linker B) 10 uM MSLXXB (MSL-C) 0.8 mM MAAD 1 mMDLP (Linker A) 10 mM GMPE

2nd layer: Ga . . . B=100,000:1, using either GaXB, GaXXB, GaXXXB, orGa(XXX)₂ (Linkers E) 28 mM GMPE

(Note: GMPE=GDPE with a monophytanyl chain instead of diphytanyl chains)

Electrodes were prepared and 2nd layer added as described in Example 4.SA was titrated into membranes containing the different types ofgramicidin. FIG. 12 shows the dependence of the SA response on linkertype and length. The response was measured by normalising the frequencyat the phase minima, i.e. calculating

(phase_(final)−phase_(initial))/phase_(final).

EXAMPLE (7)

Preparation of Bilayer Membrane

The structure of “linker lipid A” is shown in FIG. (1); the structure of“linker gramicidin B” is shown in FIG. (2); the structure of “membranespanning lipid D” is shown in FIG. (3); the structure of “membranespanning lipid C” where n=2 is shown in FIG. (3),the structure of“biotinylated gramicidin F” used is shown in FIG. (5),the structure of“biotinylated gramicidin E” used, where n=5, is shown in FIG. (4).

A glass slide or plastic support is evaporatively coated with a 50angstrom chromium adhesion layer, followed by a 2000 angstrom layer ofgold. The gold coated substrate is placed in an ethanolic solutioncontaining “linker lipid A” (300 ul of 10 mM solution in ethanol), thedisulfide of mercaptoacetic acid (150 ul of a 10 mM solution inethanol), “linker gramicidin B” (100 ul of a 0.01 mg/ml solution inethanol), “membrane spanning lipid D” (225 ul of a 1 mM solution inethanol), “membrane spanning lipid C” (22.3 ul of a 0.01 mM solution inethanol) and ethanol (50 ml). The gold coated substrate shouldpreferably be placed into this solution within five minutes ofpreparation. The gold coated substrate is left in this solution for 60minutes, and then rinsed with ethanol. The slide may then be stored inethanol, water+sodium azide (0.01% w/v), ethylene glycol, glycerol,decane, decanol or hexadecane until required. When needed, the goldcoated slide is rinsed with ethanol and is then assembled in anelectrode holder such that an electrode is defined, that for the currentexamples has an area of approximately 16 mm². Then 5 ul of a solution of1,2-di(3RS,7R,11R-phytanyl)-glycero-3-phosphocholine and1,2-di(3RS,7R,11R-phytanyl)glycerol in a 7:3 ratio, 14 mM total lipidconcentration in ethanol is added to the surface of the gold electrodeand then rinsed with two washes of 500 ul of phosphate buffered saline(PBS), leaving 100 ul PBS above the electrode surface. A counterelectrode, typically silver, is immersed in the PBS solution; and thecounter electrode and the sensing electrode are connected to animpedance bridge. A DC offset of −300 mV is applied to the sensingelectrode during the AC measurement.

EXAMPLE (8)

Preparation of Solubilised Gramicidin

EXAMPLE (8A)

A solution of “linker gramicidin E” (1 uM) and sodium dodecylsulfate (10uM) in PBS is sonicated in a bath sonicator for 20 minutes. Thissolution may be stored for at least 12 months at 4° C. Although thegramicidin with sodium dodecylsulfate is stable in aqueous solution, thegramicidin incorporates readily into sensing membranes and producesconducting ion channels. This change in conduction can be monitoredusing impedance spectroscopy.

EXAMPLE (8B)

Alternatively, a solution of “linker gramicidin F” (20 ul of 10 uM inethanol) was added to a solution of streptavidin (200 ul of 1 mg/nil inPBS+700 ul of PBS, total volume 1 ml) and mixed by vortexing for 1minute. This solution is stable for several months at 4° C.

In the absence of either SDS or streptavidin, the ability for thegramicidin to insert into the bilayer membrane is deteriorates rapidlyover one to two days. Not wishing to be bound by scientific theory, itis assumed that the gramicidin precipitates out of the aqueous solution.

EXAMPLE (9)

Preparation of a Biosensor Membrane Using Solubilised Gramicidin

EXAMPLE (9A)

To the bilayer membrane prepared in example (7) is added a solution (10ul) of solubilised gramicidin prepared in example 8A. The conductance ofthe membrane is monitored by impedance spectroscopy. This conductanceincreases as gramicidin molecules insert into the bilayer membrane.Addition of equivalent amounts of SDS or streptavidin without anygramicidin do not cause significant increases in conduction of themembrane. Prior to addition of the solubilised gramicidin the impedanceat 10 Hz was 170 kohm/16mm². After addition of the solubilisedgramicidinthe impedance was monitored until the desired level ofconductance had been achieved, (in this case an impedance of 41 kohms/16mm² at 10 Hz) the electrode well was rinsed with PBS (3×500 ul).Streptavidin (5 ul of 0.1 mg/ml in PBS) is added to the electrode well,left for three to five minutes and rinsed with PBS (3×500 ul). In thecase of a ferritin responsive sensor, biotinylated anti-ferritin Fab′ (5ul of 0.06 mg/ml in PBS) was added and after three to five minutes theelectrode well was rinsed with PBS. In the case of a thyroid stimulatinghormone (TSH) sensor a 1:1 mixture of two complementary biotinylatedanti-TSH Fab's (10 ul of 0.01 mg/ml) was added. The biotinylated Fab'swere biotinylated via the free thiol group of freshly cleaved (Fab)₂dimers. The sensor is now ready for addition of the analyte solution.Addition of a test solution of ferritin in PBS such that the final wellconcentration was 200 pM of ferritin gave an increase in impedance from41 kohms/16 mm² to 74 kohms/16 mm². The impedance spectra are shown inFIG. 14.

EXAMPLE (9B)

To the bilayer membrane prepared in example (7) is added a solution ofstreptavidin (5 ul of 0.1 mg/ml in PBS). After three to five minutes theelectrode well is rinsed with PBS and one of a complementary pair ofbiotinylated anti-TSH Fab′ (10 ul of 0.01 mg/ml in PBS) is added. After.3 to 5 minutes the electrode is rinsed with PBS and a solution ofsolubilised gramicidin as prepared in example (8B) (10 ul) is added. Theimpedance of the membrane is monitored until the desired conduction isachieved (in this case an impedance of 49 kohm/16 mm² at 10 Hz) and theelectrode is then rinsed with PBS. The other of the complementary pairof biotinylated anti-TSH Fab′ (10 ul of 0.01 mg/ml in PBS) is then addedand after three to five minutes the electrode is rinsed. The sensor nowhas the first of the complementary anti-TSH Fab′ attached to the“membrane spanning lipid C” and the second of the complementary anti-TSHFab′ attached to “linker gramicidin F”. Addition of a test solution ofTSH in PBS such that the final analyte concentration in the well was 500pM gave an increase of impedance from 49 kohm/16 mm² to 60 kohm/16 mm².The impedance spectra are shown in FIG. 15.

EXAMPLE (10)

Dry Storage of Sensor Membrane

EXAMPLE (10A)

Sensor membranes were prepared as in example (7).

The sensor membranes were then rinsed with a 0.5% (w/v) glycerol inwater solution (+0.1% sodium azide). Excess glycerol solution wasremoved such that 20 ul of the glycerol solution was left in theelectrode well assembly. The sensor membrane was then placed in achamber containing dessicant (RH in chamber was approximately 15%) andallowed to dry. The dry sensor membrane could then be stored at 15%-70%RH at room temperature for up to 1 week. It was found that when themembranes were dried from solutions with glycerol concentrations of lessthan 0.1% w/v the membranes became excessively leaky on rehydration withPBS. Thus, the impedance at 10 Hz for a freshly prepared, sealedmembrane that has not been dried was between 129-149 kohm/16 mm², whilethe impedance at 10 Hz for membranes that had been dried from 5-0.1% w/vglycerol solution was between 100-110 kohm/16 mm². Membranes that hadbeen dried from less than 0.1% w/v glycerol solution became very leakywith 10 Hz impedances of less than 45 kohm.

Impedance at 10 Hz (average of 16 e1ectrodes): Freshly prepared 5 daysat 15% RH 5% glycerol w/v 148 kohm 118 kohm 0.5% glycerol w/v 139 kohm 98 kohm 0.1% glycerol w/v 148 kohm 103 kohm 0.05% glycerol w/v 136 kohm 35 kohm

One of the advantageous properties of excess drying agent is that itprotects the bilayer lipid membrane from passing through an air/waterinterface. The air/water interface may destabilise and disrupt certainlipid bilayer structures. The glycerol coating allows for controlledrehydration of the lipid membrane without the lipid bilayer immediatelycontacting the air/water interface as the analyte solution is added, asthe rate of dissolution of the glycerol is slower than the rate ofaddition of the analyte solution.

It was found that if the sensor membrane was stored at <50% RH then onaddition of analyte solution an equilibration/rehydration periodoccurred that lasted 30-90 seconds. This equilibration is notnecessarily a problem when determining analyte concentration as it maybe subtracted using a second non-sensing differential electrode.However, it is also possible to remove this equilibration/rehydrationeffect by pre-equilibrating the dry sensor membranes for a period oftime in an atmosphere of RH of approximately 70%. Typically thispre-equilibration may be for 5-90 minutes prior to addition of analytesolution.

EXAMPLE (10B)

Sensor membranes were prepared as in example (7) except that “linkergramicidin E” was incorporated into the bilayer at lipid:gramicidinratio of 4,0000:1. The membranes were dried from 0.5% w/v glycerolsolution as described in example (10A) and were subsequently stored for5 days at room temperature at approximately 15% RH. The sensor membraneswere then rehydrated with PBS solution and streptavidin was added (5 ulof 0.1 mg/ml). The rate of increase in the impedance was measured. Aconvenient measure was the frequency at the minimum phase taken from astandard Bode plot of the phase versus time. An exponential curve(y=−ke^(t/tau)) was fitted to the response rate curve and as a measureof the rate of gating towards the streptavidin the tau value was used.The tau value is related to the analyte concentration. It was found thatover a period of five days the tau value in response to streptavidingating did not vary within experimental error. Thus:

Days of Storage Tau (s), (std. deviation) 0 71 (26) 1 79 (14) 2 56 (12)3 80 (27) 5 77 (16)

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

What is claimed is:
 1. A biosensor comprising a rehydratable membraneobtained by separating an aqueous bathing solution from a lipid membranein contact with the solution in a manner that prevents the lipidmembrane from coming into contact with an air-water interface, the lipidmembrane incorporating ionophores and optionally receptor molecules andhaving a conductivity that is dependent on the presence or absence of ananalyte, the rehydratable membrane being such that on rehydration, thelipid membrane and any receptor molecules retain their function,structure and activity.
 2. A biosensor according to claim 1, wherein theaqueous bathing solution is removed by a drying method selected from thegroup consisting of lyophilysation, evaporation and evaporation overcontrolled humidity.
 3. A biosensor according to claim 1, wherein theaqueous bathing solution is replaced by a water-replacing substance. 4.A biosensor according to claim 3, wherein the water replacing substanceis selected from the group consisting of bovine serum albumin, serum,fish gelatin, non-fat dry milk powder, casein, glycerol, ethyleneglycol, diethylene glycol, polyethylene glycol, trehalose, xylose,glucose, sucrose, dextrose, dextran and ficoll.
 5. A biosensor accordingto claim 4, wherein the water replacing substance is selected from thegroup consisting of glycerol, sucrose, dextran and trehalose.
 6. Abiosensor according to claim 3 further comprising the step of subjectingthe lipid membrane to a drying method selected from the group consistingof lyophilysation, evaporation and evaporation over controlled humidity.7. A biosensor according to claim 3, wherein the water-replacingsubstance is selected from the group consisting of protein, lowmolecular weight diols, low molecular weight triols, polyethyleneglycol, low molecular weight sugars, polymeric peptides,polyemelectrolyte and combinations thereof.
 8. A biosensor according toclaim 3, wherein the water-replacing substance further provides aspreading layer for a sample or component thereof.
 9. A biosensoraccording to claim 3, wherein the water-replacing substance further actsas a filter against a sample or component thereof selected from thegroup consisting of specific cells, bacteria, viruses and classes ofmolecules.
 10. A biosensor according to claim 9, wherein thewater-replacing substance further acts as a filter against largemolecular weight proteins.
 11. A biosensor according to claim 3, whereinthe water-replacing substance further provides a reservoir for specificdisplacement reagents that complete with small analytes on proteins towhich they are bound.
 12. A biosensor according to claim 3, wherein thewater-replacing substance is covalently bound to membrane components.13. A biosensor according to claim 12, wherein membrane components aremembrane spanning lipids.
 14. A lipid based biosensor formed byrehydrating the membrane of a biosensor according to claim
 1. 15. Abiosensor comprising a rehydratable membrane formed by removing anaqueous solution with which a lipid membrane has been in contact, thelipid membrane incorporating ionophores and optionally receptormolecules and having a conductivity that is dependent on the presence orabsence of an analyte, wherein the rehydratable membrane is such that onrehydration, the lipid membrane and any receptor molecules retain theirfunction, structure and activity.
 16. A biosensor suitable for drystorage comprising a rehydratable membrane, said membrane comprising:(a) a lipid membrane incorporating ionophores and optionally receptormolecules and having a conductivity that is dependent on the presence orabsence of an analyte; and (b) a water-replacing substance.
 17. Abiosensor according to claim 16, wherein the water-replacing substanceis selected from the group consisting of protein, low molecular weightdiols, low molecular weight triols, polyethylene glycol, low molecularweight sugars, polymeric peptides, polyemelectrolyte and combinationsthereof.
 18. A biosensor according to claim 16, wherein the waterreplacing substance is selected from the group consisting of bovineserum albumin, serum, fish gelatin, non-fat dry milk powder, casein,glycerol, ethylene glycol, diethylene glycol, polyethylene glycol,trehalose, xylose, glucose, sucrose, dextrose, dextran and ficoll.
 19. Abiosensor according to claim 16, wherein the water replacing substanceis selected from the group consisting of glycerol, sucrose, dextran andtrehalose.
 20. A biosensor according to claim 16, wherein therehydratable membrane is formed by contacting the lipid membrane withthe water replacing substance followed by drying of the lipid membrane.21. A biosensor according to claim 20, wherein the lipid membrane iscontacted with an aqueous solution of the water replacing substance. 22.A biosensor according to claim 16, wherein the water-replacing substanceis covalently bound to the lipid membrane.
 23. A biosensor according toclaim 22, wherein the water-replacing substance is covalently bound tomembrane components.
 24. A biosensor according to claim 23, whereinmembrane components are membrane spanning lipids.
 25. A biosensorcomprising a rehydratable lipid membrane which has been prepared by aprocess comprising the steps of: (i) mixing an aqueous solutioncontaining membrane components, ionophores and optionally receptormolecules; (ii) allowing the membrane to be formed in the aqueoussolution, the conductivity of the formed membrane being dependent on thepresence or absence of an analyte; and (iii) separating the aqueoussolution from the membrane in a manner such that the membrane does notcome into contact with an air-water interface, wherein the rehydratablelipid membrane is such that on rehydration, the lipid membrane and anyreceptor molecules retain their function, structure and activity.